The steroid/thyroid hormone receptors comprise a superfamily of ligand-dependent transcription factors that play a crucial role in mediating changes in cell fate and function (Evans, R. M., Science 240:889–895 (1988)). The receptors transduce extracellular hormonal signals to target genes that contain specific enhancer sequences referred to as hormone response elements (HREs) Evans, (1988); Green and Chambon, Trends Genet. 4:309–314 (1988); Yamamoto, K. R., Annu. Rev. Genet. 19:209–252 (1985)). Each receptor recognizes its own HRE, assuring that a distinct response is triggered by each hormonal signal. Together the collection of related transcription factors and their cognate response elements provides a unique opportunity to control gene expression.
The DNA binding domain of each member of the steroid/thyroid hormone superfamily of receptors has 66–68 amino acids. Twenty of these, including nine cysteines, are conserved throughout the family. The modular structure of members of this receptor superfamily allows the exchange of homologous domains between receptors to create functional chimeras. This strategy was used to demonstrate that the DNA binding domain is solely responsible for the specific recognition of the HRE in vivo (Green and Chambon, Nature 325:75–78 (1987); Giguere et al., Nature 330:624–629 (1987); Petkovich et al., Nature 330:444–450 (1987); Kumar et al., Cell 51:941–951 (1987); Umesono et al., Nature 336:262–265 (1988); Thompson and Evans, Proc. Natl. Acad. Sci. U.S.A. 86:3494–3498 (1989) and in vitro (Kumar and Chambon, Cell 55:145–156 (1988)). By analogy with the proposed structure for Xenopus transcription factor IIIA (Miller et al., EMBO J. 4:1609–1614 (1985)), the invariant cysteines are thought to form two “zinc fingers” that mediate the DNA binding function (Hollenberg and Evans, Cell 55:899–906 (1988)). Involvement of these cysteines in Zn(II) coordination is supported by extended X-ray absorption fine structure (Freedman et al., Nature 334:543–546 (1988)), and by the effect of point mutagenesis experiments on DNA binding (Hollenberg and Evans, (1988)); Severne et al., EMBO J. 7:2503–2508 (1988)).
The HREs are in fact structurally related but functionally distinct. The glucocorticoid receptor response element (GRE), estrogen receptor response element (ERE), and thyroid hormone receptor response element (TRE) have been characterized in detail. These particular response elements have been found to have a palindromic pair of hexameric “half-sites” (Evans, (1988); Green and Chambon, (1988)). With optimized pseudo- or consensus response elements, only two nucleotides per half-site differ between GRE and ERE (Klock et al., Nature 329:734–736 (1987)). On the other hand, EREs and TREs have identical half-sites but the number of nucleotide spacers between the two half sites is different (Glass et al., Cell 54:313–323 (1988)).
In contrast to response elements having the palindromic sequence motif, the following hormone receptors typically recognize response elements having two half-sites in a direct-repeat (DR) sequence motif: RXR, RAR, COUP-TF, PPAR, and the like (see, e.g., Mangelsdorf et al., The Retinoids: Biology, Chemistry, and Medicine, 2nd Edition, Raven Press, Ltd., New York, 1994, Chapter 8). Thus at least three distinct means are used to achieve HRE diversity: 1) binding site specificity for a particular half-site; 2) nucleotide spacing between the two half-sites; and 3) the orientation of the half-sites to one another.
In insect systems, a pulse of the steroid hormone ecdysone triggers metamorphosis in Drosophila melanogaster showing genomic effects, such as chromosomal puffing, within minutes of hormone addition. Mediating this response in insects is the functional ecdysone receptor, a heterodimer of the ecdysone receptor (EcR) and the product of the ultraspiracle gene (USP) (Yao et al., Nature 366:476–479 (1993); and Yao et al., Cell 71:63–72 (1992)). Responsiveness to an insect ecdysteroid can be recreated in cultured mammalian cells by co-transfection of EcR, USP, an ecdysone responsive reporter, and treatment with ecdysone or the synthetic analog muristerone A.
The ability to manage the expression of genes introduced into mammalian cells and animals would further advance many areas of biology and medicine. For instance, methods that allow the intentional manipulation of gene expression would facilitate the analysis of genes whose gene products cannot be tolerated constitutively or at certain stages of development. Such methods would also be valuable for clinical applications such as gene therapy protocols, where the expression of a therapeutic gene must be regulated in accordance with the needs of the patient (Saez et al., Curr. Opin. Biotechnol. 8:608–616(1997)). However, to be of broad benefit, gene regulation techniques must allow for rapid, robust and precise induction/repression of gene activity. Precise control of gene expression is an invaluable tool in studying, manipulating and controlling development and other physiological processes.
Early designs to direct gene expression in mammals were based on endogenous elements, such as cytokine response elements or heat-shock proteins. Due to a high level of basal expression in the uninduced state, and pleiotropic effects brought about by general inducing agents, these systems lack the specificity required to regulate genes in mammalian cells and organisms. (Saez et al., supra)
As another means for controlling gene expression in a mammalian system, an inducible tetracycline regulated system has been devised and utilized in transgenic mice, whereby gene activity is induced in the absence of the antibiotic and repressed in its presence (see, e.g, Gossen et al., Proc. Natl. Acad. Sci. 89:5547–5551 (1992); Gossen et al., TIBS 18:471–475 (1993); Furth et al., Proc. Natl. Acad. Sci. 91:9302–9306 (1994); and Shockett et al., Proc. Natl. Acad. Sci. 92:6522–6526 (1995)). However, problems were noticed during the development of this system including toxicity of the tetracycline transactivator protein, the requirement for continuous treatment of tetracycline to repress expression, and the slow clearance of antibiotic from bone which interferes with quick and precise induction. While this system has been improved by the recent identification of a mutant tetracycline repressor which acts conversely as an inducible activator, the pharmacokinetics of tetracycline may hinder its use during development when a precise and efficient “on-off” switch is essential (Gossen et al., Science 268:1766–1769 (1995)).
Another approach to regulate gene expression relies on induction of protein dimerization, a method derived from studies on the mechanism of action of immunosuppressive agents (Spencer, D M, Trends Genet, 12:181–187 (1996)). Compounds such as FK506 and cyclosporin A (CsA) subdue the immune response by binding with high affinity to the immunophilins FKBP12 and cyclophilin (CyP), respectively. Using a synthetic homodimer of FK506 (called FK10102), a general strategy was devised to bring together any two peptides, by endowing them with the domain of FKBP12 to which FK506 binds. By chemically linking FK506 and CsA, a heterodimer molecule that can selectively connect two different immunophilin domains and their respectively attached peptides was also generated.
The resulting heterodimerizer, FKCsA, has been used to reconstitute a functional transcription factor by joining a GAL4 DNA-binding domain fused to FKBP12, and the transactivation moiety of VP16 bound to CyP. In cells expressing these chimeric proteins, the expression of a promoter containing GAL4 binding sites was strongly stimulated in the presence of this “chemical inducer of dimerization” (CID) FKCsA.
The immunosuppressive drug rapamycin is a natural heterodimerizer that complexes with FKBP12 and FKBP12-rapamycin-associated protein (FRAP). A new inducible system based on rapamycin builds on the modularity of mammalian transcription factors and the heterodimerizing properties of this drug (Rivera et al., Nature Med. 2:1028–1032 (1996)). Unfortunately, the attractive pharmacokinetics of this drug are compromised by its effects on the immune system, i.e., rapamycin is incapable of regulating gene expression at doses that are not immunosuppressive.
Two gene control systems based on components of mammalian steroid hormone receptors have recently been developed (Wang et al., Proc. Natl. Acad. Sci. U.S.A. 91:8180–8184 (1994); Delort and Capecchi, Num. Gene Ther. 7:809–820(1996)). Created independently, these two steroid-based methods are nonetheless virtually identical, i.e, both methods combine a truncated form of the progesterone receptor hormone-binding domain with a yeast GAL4 DNA-binding moiety, and the transactivation domain of the VP16 protein. The mutated progesterone receptor moiety fails to bind progesterone but it retains the ability to bind the progesterone and glucocortoid antagonist mifepristone (RU486), such that in the presence of RU486, the fusion protein (called either GLVP or TAXI) activates transcription through a multimer of the GAL4 DNA-binding site placed upstream of a minimal promoter.
Although these systems represent an improvement over previous hormone based designs, their performance in cells remains poor. In transient and stable transfections of various cell types, a high level of basal activity dampens the level of inducibility of the above-described approaches. Moreover, since RU486 is an abortifacient, these systems are not likely to be useful for developmental studies. Furthermore, the utility of these approaches for long-term protocols is a concern as the response to RU486 diminishes over time. Experiments with these systems have also hinted at the possibility that these chimeric proteins may interfere with endogenous factors, an observation that would explain why it was difficult to generate transgenics that express these proteins. In spite of these issues, an important advantage of steroid-based systems is that they appear to have more favorable kinetics than tetracycline systems, i.e., lipophilic hormones employed by steroid-based systems are quickly metabolized and have short half-lives in vivo.
Accordingly, there remains a need in the art for improved methods to precisely modulate the expression of exogenous genes in mammalian subjects, as well as expanding the range of formulations which can be used to modulate such systems.